FMR1 deletion in rats induces hyperactivity with no changes in striatal dopamine transporter availability

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder emerging in early life characterized by impairments in social interaction, poor verbal and non-verbal communication, and repetitive patterns of behaviors. Among the best-known genetic risk factors for ASD, there are mutations causing the loss of the Fragile X Messenger Ribonucleoprotein 1 (FMRP) leading to Fragile X syndrome (FXS), a common form of inherited intellectual disability and the leading monogenic cause of ASD. Being a pivotal regulator of motor activity, motivation, attention, and reward processing, dopaminergic neurotransmission has a key role in several neuropsychiatric disorders, including ASD. Fmr1 Δexon 8 rats have been validated as a genetic model of ASD based on FMR1 deletion, and they are also a rat model of FXS. Here, we performed behavioral, biochemical and in vivo SPECT neuroimaging experiments to investigate whether Fmr1 Δexon 8 rats display ASD-like repetitive behaviors associated with changes in striatal dopamine transporter (DAT) availability assessed through in vivo SPECT neuroimaging. At the behavioral level, Fmr1 Δexon 8 rats displayed hyperactivity in the open field test in the absence of repetitive behaviors in the hole board test. However, these behavioral alterations were not associated with changes in striatal DAT availability as assessed by non-invasive in vivo SPECT and Western blot analyses.

SPECT System. The High-Resolution Imaging System (HiRIS2) for preclinical studies has been used to quantify the dopamine transporter (DAT) binding in the brain of the Fmr1-Δ exon 8 and WT rats using [ 123 I]FP-CIT as radioligand. The dedicated SPECT scanner consists of two detection heads housed in a rotating gantry. The experimental subject is placed on an animal bed that can move axially with respect to the rotation plane. The mechanics are designed to allow the necessary movements of the detectors for the application of the Super Spatial Resolution (SSR) technique which could be applied to improve the effective spatial resolution achievable, thus representing a key element for the study of very small brain structures 38 . To this purpose, multi-degree freedom carriages are used to obtain a fine alignment both linear and planar of the detectors. Specifically, each HiRIS2 head is based on an H13700 Hamamatsu PSPMT coupled to a CRY018 pixelated scintillator and a lowenergy tungsten collimator with parallel square holes. Finally, to obtain a 3D SPECT imaging, the two detectors were mounted in opposition at 180° from each other.
Behavioral procedures. The animals were habituated to the experimental room before testing. To minimize stress responses and to allow the animals to familiarize with the operator, rats were extensively handled for few consecutive days before testing by the same operator who performed the test. Notably, the scoring-designed operator was not the same operator who manipulated the animals and performed the test, and was unaware of animals' genotype; in other words, scoring was done in blind conditions.
Hole board test. The test was performed in a sound-attenuated chamber under dim light conditions, as previously described [41][42][43][44] . The apparatus consisted of a grey square metal table (40 × 40 × 10 cm; l × w × h) with 16 evenly spaced holes (4 cm in diameter), inserted in a Plexiglas arena (40 × 40 × 60 cm; l × w × h). Each rat was individually placed in the apparatus for 5 min. Each session was recorded with a camera positioned above the apparatus for subsequent behavioral analysis performed using the Observer 3.0 software (Noldus Information  www.nature.com/scientificreports/ Technology, NL). Dipping behavior was scored as the number of times an animal inserted its head into a hole at least up to the eye level.
Open field test. The test was performed as previously described 45 . The apparatus consisted of a Plexiglas arena  45 × 45 cm, illuminated by fluorescent bulbs at a height of 2 m above the floor of the open field apparatus (light  intensity of 30 lx). The floor was cleaned between each trial to avoid olfactory clues. Each animal was transferred to the open field facing a corner and was allowed to freely explore the experimental arena for 15 min. The locomotor activity was scored as follows 43 : a grid, dividing the arena into equally sized squares, was projected over the recordings, and the number of line crossings made by the animal (i.e., the frequency of the animal's passage from one section of the grid to another) was recorded using the Observer 3.0 software (Noldus Information Technology, NL). Notably, the crossing was counted at the time when the animal passed from one section to another with all four paws 46 . Spontaneous rearing behaviors, in which rodents stand on their hind legs with the intention of exploring, were also counted during the 15-min test period. We defined two forms of rearing: unsupported rearing (in which the animal rears without contacting the walls of the arena) and wall rearing (in which the animal rears against the walls of the arena). To evaluate thigmotaxis (i.e., the animal remains closely in proximity to the walls of the open field), the time spent at periphery and at center of the open field was also measured and reported as percentage of the total time.
Elevated Plus Maze test. The elevated plus maze apparatus comprised two open (50 × 10 × 40 cm; l × w × h) and two closed arms (50 × 10 × 40 cm; l × w × h) that extended from a common central platform (10 × 10 cm). The test was performed as previously described 47,48 . Rats were individually placed on the central platform of the maze for 5 min. Each 5-min session was recorded with a camera positioned above the apparatus for subsequent behavioral analysis carried out an observer, unaware of animal treatment, using the Observer 3.0 software (Noldus Information Technology, NL). The following parameters were analyzed: Neuroimaging studies. SPECT measurements of DAT binding sites were performed with the HiRIS2 SPECT system as previously described 39 . Animals were pre-treated by oral gavage with 10 μl of Lugol's solution, a thyroid blocking agent, 1 h prior to the administration of the radiopharmaceutical. This will decrease the radiation dose to the thyroid and preclude possible adverse effects on the gland, while also allowing better brain imaging by concentrating iodine accumulation on the targeted area [49][50][51] . Then, the animals were anesthetized using isoflurane (IsoFlo, Zoetis, UK) at a concentration of 3% for induction and 2% for maintenance. In vivo DAT binding was measured using Methyl (3S,4S,5R)-8-(3-fluoropropyl)-3-(4-iodophenyl)-8-azabicyclo[3.2.1] octane-4-carboxylate([ 123 I]FP-CIT, DaTSCAN ® ) as radioligand, since it is widely used to assess the pre-synaptic striatal uptake in the basal ganglia of the rat brain [52][53][54][55] . A dose of 37 ± 4 MBq in 1,25 ml DaTSCAN (GE Healthcare, DE) was administered into the lateral tail vein. Imaging measurements were started 2 h after radioligand administration when the equilibrium post-injection of [ 123 I]FP-CIT binding is reached, with the ratio of specific to non-specific striatal uptake remaining stable over the following 4 h 56 .
Generally, in small animal imaging studies intravenous injection of [ 123 I]FP-CIT (DaTSCAN) has been used to visualize both the DAT and the serotonin transporters (SERT) 54 . However, DaTSCAN injection results in high radioactivity accumulation in the striatum. In comparison, less pronounced uptake has been observed in brain areas with a high density of serotonergic uptake sites, such as the midbrain 56 . Therefore, when assessing striatal DAT function in vivo in laboratory animals, only striatal signal is usually considered, both because of the limited sensitivity and resolution of the available instrumentation and because of the possibility of seeing elevated extrastriatal signals in the midbrain also due to the binding of DaTSCAN to the SERT 57,58 .
Both planar and SPECT acquisitions were performed. Planar images were obtained by 30 min acquisitions. Whereas, each SPECT scan was carried out by collecting 48 angular projections over an arc of 360° (24 steps of 7.5°). Since the SSR method was applied, two images per angular position were acquired. Subsequently, the total scan time was 48 min. Since the CT module is not implemented on the HiRIS2 prototype, we could perform a CT scan on each rat's brain using a U-CT preclinical system (MILabs B.V., NL). SPECT reconstruction was performed with the iterative ordered-subset-expectation-maximization (OSEM) algorithm using a priori knowledge of the CT data providing a precise localization of the radiotracer uptake.
Computed Tomography (CT) imaging. For coregistration of SPECT functional data to tomographic ones, three-dimensional morphological volume measurements were acquired with a micro-CT scanner having 0.08 mm isotropic resolution. The rats' brains of both Fmr1-Δ exon 8 rats and their WT control group were scanned using a MILabs 3D optical CT scanner (MILabs B.V., NL). CT scanner settings were identical for all scans. Each rat brain was 3-dimensionally (3D) micro-CT acquired at 80-μm voxel size resolution using the following settings: 720 steps of 0,5°; exposure 40 ms; voltage 50 kV; current, 0.43 mA, yielding a total scan time of about 2,5 min. The reconstruction output was the Neuroimaging Informatics Technology Initiative (NIftI) file type, which is a format commonly used in preclinical nuclear imaging informatics. CT scan reconstructions were performed using filtered back-projection (FBP) for the registration to SPECT scans. Finally, coregistered images were processed using Mango-Multi-image Analysis GUI (Research Imaging Institute, UTHSCSA) 59 60 image software. Axial, coronal, and sagittal views and SPECT slices co-registered with the anatomic reference provided by the CT are shown in Fig. 5.
Histology. At the end of the experiments, the animals were sacrificed, and brain samples were quickly collected and fixed in formalin 10%. For hematoxylin (Sigma-Aldrich, cat. MHS16) and eosin (Sigma-Aldrich, cat. 109,844) histology assay samples were paraffin embedded. Microtome-sectioning was conducted to generate 8-μm sections that were deparaffinized and rehydrated. For histological analysis, standard hematoxylin and eosin staining was performed to evaluate significant changes in the gross tissue organization of the dorsal striatum between Fmr1-Δ exon 8 and WT rats, and bright-field images were acquired using an inverted microscope equipped with 6 × and 40 × objective (Leica Microdissector-LMD 7000, camera Leica DFC310 FX). The histological and immunostaining assays were performed on dorsal striatum to further support the results achieved by SPECT analysis.
Immunohistochemistry. Immunohistochemistry was conducted on deparaffinized and rehydrated 8-μm Evaluation of Neuroimaging studies. Regarding planar acquisitions, for each rat, SPECT and CT images were coregistered within the aim of the Paxinos standard rat brain MRI46 provided by the Neuroimaging Tools & Resources Collaboratory (NITRC Image Repository (NITRC-IR)-www. nitrc. org). At this point, image processing and analysis were performed through a semi-quantitative evaluation of the region of interest (ROI) on brain-activated areas. In particular, as shown in Fig. 3, imaging data were assessed by defining an area comprising each striatum, and another corresponding to the cerebellum region. Thus, maximum striatal count rates (counts/pixel), as well as cerebellar reference count rates (counts/pixel), were determined. Left and right striatal counts rates were averaged. Semi-quantitative measures for the quantification of the binding potential were assessed as ratios between the striatal specific uptake and the non-specific cerebellum uptake 61 . Particularly, the striatal specific binding ratio (SBR STR ) was calculated as: where C STR and C CB are, respectively, the mean count in the striatal and in the cerebellum ROI. Instead of the volumetric reconstructions, three-dimensional CT and all reconstructed SPECT studies were transferred to a dual Xeon processor (Intel Corporation, USA) workstation for images analysis and 3D rendering (Fig. 5). Statistical analysis. Behavioral analysis. Data are expressed as mean ± standard error of the mean (S.E.M.). To assess the effects of the genotype on behavioral parameters, data were analyzed with Student's ttests. Sample size (n) is indicated in the figure legends and was based on our previous experiments and power analysis performed with the software G*Power. Potential outliers within each data set were calculated using GraphPad Prism 8 software (Grubbs' method). A trained observer who was unaware of the treatments assessed all behavioral tests that were scored using the Observer 3.0 software (Noldus Information Technology, NL).

Western blot analysis.
DAT imaging analysis. The measured SBR STR values of the two groups were analyzed with Student's t-tests and expressed as mean ± S.E.M. Data analysis was performed using GraphPad Prism 8 software.
Western blot analysis. The statistical differences between the two groups were analyzed with Student's t-tests and relative values expressed as mean ± S.E.M. Data analysis was performed using GraphPad Prism 8 software.
Ethical approval. This study was performed and reported in compliance with the ARRIVE guidelines 40,62 .
All applicable Institutional and National guidelines for the care and use of animals were followed. The protocol was approved by the Italian Ministry of Health (Rome, Italy; Authorization N° 849/2020-PR).  Supplementary Fig. 1 (C and G)). Conversely, both juvenile and adult Fmr1-Δ exon 8 rats displayed hyperactivity in the elevated plus maze test as they showed an increased number of total arm entries when compared to their WT controls (PND 35-40: t = 3.64, p < 0.001, df = 33; PND 80-85: t = 2.87, p < 0.01, df = 34; Supplementary Fig. 1  (D and H)). This further confirms the hyperlocomotion displayed by Fmr1-Δ exon 8 rats across development.

Behavioral studies. Stereotypic behavior and locomotor activity in
Neuroimaging studies. Figure 3 shows the characteristic planar images of [ 123 I]FP-CIT uptake of two WT rats (panel A) and two Fmr1-Δ exon 8 (panel B) rats, respectively. Radioactivity accumulations are clearly visible in the striatum. Moreover, the image also highlights the ROIs related to both striatum and cerebellum for each experimental group. SBR STR analysis reveals that in the WT group the striatal specific binding ratio was 0.608 ± 0.087 (mean ± S.E.M.), whereas for the Fmr1-Δ exon 8 rats the value was 0.658 ± 0.034 (mean ± S.E.M.). As a result, the unpaired t-test revealed no significant between-group differences (t = 0.539, p = 0.604, df = 8, Fig. 3C). Since the images also showed enhanced DAT levels in the midbrain, its specific binding ratio have been assessed. Namely, the midbrain specific binding ratio was 0.332 ± 0.135 (mean ± S.E.M.) in the WT group, and 0.403 ± 0.034 (mean ± S.E.M.) in Fmr1-Δ exon 8 rats. As with striatal uptake, the unpaired t-test revealed no significant between-group differences (t = 0.716, p = 0.548, df = 2). An example of the characteristic distribution of DAT in the striatum in both experimental groups, WT (top) and Fmr1-Δ exon 8 (bottom), is also shown through an orthogonal view of the SSR SPECT OSEM reconstruction restricted to CT brain data (Fig. 4). Moreover, the DAT binding distribution with respect to the correlated CT brain volume obtained from a 3D rendering of the dataset is shown in Fig. 5 (WT on top and Fmr1-Δ exon 8 on bottom images). An initial visual analysis of the images for both groups does not show a difference in the number and extent of radiopharmaceutical uptake, which is overall normal for both nigrostriatal systems. This assessment is corroborated by semi-quantitative SBR STR analysis, which reveals no substantial differences between the two groups. www.nature.com/scientificreports/ Immunofluorescence and immunohistochemistry analysis. Hematoxylin and eosin (H&E) staining was performed to visualize possible gross histopathological changes in the dorsal striatum of Fmr1-Δ exon 8 rats as compared to their WT controls. We found that the structure of the Fmr1-Δ exon 8 and WT rat brain tissues analyzed by H&E histology (Fig. 6A and B for WT rats, D and E for Fmr1-Δ exon 8 rats) was comparable at the level of the dorsal striatum (marked with a black arrow), suggesting no changes in the gross tissue organization of this brain region between Fmr1-Δ exon 8 and WT rats. Moreover, immunohistochemical staining relative to DAT expression (highlighted as a dark brown dye) in the dorsal striatum (Fig. 6C for WT and F for Fmr1-Δ exon 8 rats) showed no qualitative differences between genotypes as also confirmed by immunofluorescence in the striatal DA axonal projections (Fig. 6G, H and I for WT and J, K and L for Fmr1-Δ exon 8 rats). These findings strengthen our neuroimaging results revealing no differences in DAT between genotypes.
Western blot analysis. To estimate the protein expression level of DAT in the striatum of Fmr1-Δ exon 8 rats, we performed a western blotting analysis in both the dorsal (Fig. 7A and B) and ventral striatum ( Fig. 7C and D). In line with SPECT and immunohistochemical findings, the DAT protein levels in both the dorsal (t = 1.02, df = 6, p = n.s.) and ventral (t = 0.81; df = 6; p = n.s.) striatum did not differ between WT and Fmr1-Δ exon 8 animals. Overall, these data confirm that FMRP deficiency does not induce a dysregulation in striatal DAT expression and that no regional (dorsal vs. ventral striatum) differences in DAT expression underlie the dysfunctional motor behavior displayed by Fmr1-Δ exon 8 rats. Original uncropped membranes are shown in Supplementary Fig. 2 (A and B).

Discussion
In the present work, we combined behavioral, biochemical and in vivo neuroimaging analyses to investigate the role of striatal DAT expression in the dysfunctional motor behavior of Fmr1-Δ exon 8 rats, a genetic animal model of ASD and a rat model of FXS. Since FXS patients often show hyperactivity and repetitive patterns of behavior 3 , we here tested Fmr1-Δ exon 8 rats in two behavioral tasks aimed to reveal possible stereotyped/repetitive behaviors and exploratory/locomotor   www.nature.com/scientificreports/ Fmr1-Δ exon 8 rats did not display anxiety-like behaviors in this task. Conversely, both juvenile and adult Fmr1-Δ exon 8 rats showed an increased number of total entries in the arms of the elevated plus maze when compared to WT controls, thus confirming the hyperlocomotion displayed by Fmr1-Δ exon 8 rats across development. In line with these results, the extensively characterized Fmr1-KO mouse model of FXS has been shown to display motor alterations and hyperactivity [63][64][65][66][67] , although normal locomotor activity has also been also reported in rat models of FXS 68,69 . Conversely, we found that Fmr1-Δ exon 8 rats did not show stereotypic/repetitive behaviors in the hole-board test, as the number of head dippings did not differ from their WT controls. Depending on the animal strain and the behavioral task used, some studies 66,70,71 but not others 63,69,72 reported stereotyped behaviors in both rat and mouse models of FXS. Thus, we cannot exclude that Fmr1-Δ exon 8 rats would show repetitive patterns of behavior if different tasks or experimental protocols were used. Evidence for an involvement of dopaminergic neurotransmission in ASD arises from neuroimaging, genetic and pharmacological studies in individuals with autism but also from preclinical research performed in rodent models of ASD 73 . Over the years, techniques as SPECT/PET have been employed enabling imaging of the dopaminergic system in different psychiatric and neurological disorders. Such methods are often based on the assessment of DAT density as a marker for dopaminergic neuron integrity 55,[74][75][76] . Generally, a radiolabel such as 123 I-FP-CIT (DaTSCAN) is used since it is able to bind with high affinity to the presynaptic DAT located on axon terminals in the striatum. In the clinical practice, SPECT with 123 I-FP-CIT can be reported as normal or abnormal through the semi-quantitative measurement of the 123 I-FP-CIT signal uptake in the DAT 55 . Therefore, imaging with specific DA-related tracers represents a valuable tool to evaluate the status of presynaptic nigrostriatal terminals. In particular, the radiotracer DaTSCAN has become part of the diagnostic guidelines for α-synucleinopathies (e.g., Parkinsonian Syndromes, multiple system atrophy, and dementia with Lewy bodies), being approved by the most competent international authorities (i.e., FDA and EMA) 77,78 . From a methodological point of view, SPECT images might be exploited to determine to what extent this tracer is accumulated in the striatum compared to the background signal. As a result, reduced DAT striatal binding is therefore suggested to depict reduced DAT availability which in turn reflects striatal dopaminergic deficit 79 . PET imaging showed increased DAT binding in the orbitofrontal cortex of high-functioning adults with ASD 80 , although a SPECT study in children with autism showed no changes in DAT binding 81 . Interestingly, a study that examined striatal dopamine functioning during monetary incentive processing in ASD patients and controls using simultaneous PET and fMRI reported impaired phasic DA release to rewards in the striatum of patients with autism 82 . While these clinical findings support the involvement of functional changes in dopaminergic neurotransmission in ASD, differences in experimental procedures and heterogeneity in the patient populations also lead to conflicting results across studies and warrant further investigation.
Here, we took advantage from a recently developed innovative SPECT system for imaging in laboratory rodents 39 to investigate whether the hyperactivity displayed by Fmr1-Δ exon 8 rats was accompanied by changes in striatal DAT availability. By performing scintigraphic SPECT analyses in vivo using 123 I-FP-CIT as radiolabel, www.nature.com/scientificreports/ we found comparable DAT availability in the striatum of Fmr1-Δ exon 8 rats and WT controls, suggesting that no changes in striatal DAT expression occurred in Fmr1-Δ exon 8 rats. Based on these results, it is tempting to speculate that the altered locomotor activity (as expressed by the increased number of crossings in the open field and the increased total entries in the elevated plus maze) we consistently observed in Fmr1-Δ exon 8 rats along development might not be attributable to an alteration of striatal DAT availability. This evidence is also corroborated by immunohistochemistry and immunofluorescence analyses, since our results showed no difference in DAT expression between WT and Fmr1-Δ exon 8 rats in the dorsal striatum. To evaluate for a possible different contribution of the two distinct districts of the striatum (i.e., dorsal and ventral), we performed Western blot analysis of DAT protein levels: notably, no significant differences were found between genotypes, indicating that DAT protein levels were preserved in both dorsal and ventral striatum of Fmr1-Δ exon 8 rats. Despite these consistent results, we cannot exclude that pharmacological manipulation of the dopaminergic system (e.g., administration of D 1 and D 2 agonist/antagonist, cocaine, or blockers of other transporters) could have an impact on the altered   99 . Based on these (apparent) contradictory findings, we should consider the hypothesis that specific changes in DA signaling may differentially contribute to ASD pathophysiology and consequently may (not) account for the full spectrum of ASD-related behavioral manifestations 73 . For instance, it has been shown that activation of D 2 expressing neurons in the ventral striatum reduced running and locomotion in mice, while D 2 expressing neuron inhibition had opposite effects 100 ; moreover, FMRP seems to be involved in D 1 -mediated neuroplasticity in the prefrontal cortex [101][102][103] . Besides, DA receptors are differentially integrated in cortical circuit components subserving distinct aspects of cognitive control, including relaying motor commands 104 . This will undoubtedly contribute to clarify the cellular (D 1 vs. D 2 ) and regional (dorsal vs. ventral striatum, prefrontal cortex, cerebellum) specificity of DA pathways in mediating motor dysfunctions in Fmr1-Δ exon 8 rats. www.nature.com/scientificreports/ As referring to the SPECT methodology, it is important to clarify that FP-CIT is not a substrate for the transporter, hence imaging analysis only provides DAT expression. Accordingly, it is possible that alterations in the dopaminergic system contribute to the observed hyperactivity phenotype through one of the following mechanisms: (i) altered trafficking or catalytic activity of DAT; (ii) altered synthesis, packaging, or release of DA; (iii) altered sensitivity of DA receptors 105 . These hypotheses warrant further investigation in a follow-up of this study.
Overall, our results showed that Fmr1-Δ exon 8 rats displayed hyperactivity in the open field and in the elevated plus maze tests, in the absence of repetitive behaviors in the hole board test, with no changes in striatal DAT availability as assessed by in vivo SPECT imaging and Western blot experiments. This study supports a preservation of striatal DAT availability following FMR1 deletion in rats and confirms that in vivo SPECT imaging paralleled by behavioral observation represents a useful tool to non-invasively investigate variations in neurotransmitter activity in neurodevelopmental disorders. Since sex-dependent differences in preclinical models of ASD have been documented 106 , the inclusion of both male and female animals should be considered in future studies.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. www.nature.com/scientificreports/